Microarray system with improved sequence specificity

ABSTRACT

The invention provides a novel array method for nucleic acid sequence detection with improved specificity which allows for detection of genetic variation, from simple SNPs (where the variation occurs at a fixed position and is of limited allelic number) to more complex sequence variation patterns (such as with multigene families or multiple genetic strains of an organism where the sequence variation between the individual members is neither fixed nor consistent). The array is comprised of short, synthetic oligonucleotide probes attached to a solid surface which are hybridized to single-stranded targets. Single stranded targets can be produced using a method that employs primers modified on the 5′ end to prohibit degradation by a 5′-exonuclease that is introduced to degrade the unprotected strand. The invention further provides for printing buffers/solutions for the immobilization of oligonucleotide probes to an array surface. The invention also provides hybridization and wash buffers and conditions to maximize hybridization specificity and signal intensity, and reduce hybridization times.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/576,076, filed Dec. 18, 2014, which is a divisional of U.S. patentapplication Ser. No. 13/911,971, now U.S. Pat. No. 8,945,928, issuedFeb. 3, 2015, which is a continuation of U.S. patent application Ser.No. 13/650,087 filed Oct. 11, 2012, now abandoned, which is acontinuation of U.S. application Ser. No. 13/294,792 filed Nov. 11,2011, now abandoned, which is a divisional of U.S. patent applicationSer. No. 12/190,446, filed Aug. 12, 2008, now U.S. Pat. No. 8,067,164,issued Nov. 29, 2011, which claims the benefit of U.S. ProvisionalApplication No. 60/955,384, filed Aug. 12, 2007. These applications areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

Not applicable.

SEQUENCE LISTING

The sequence listing is filed with the application in electronic formatonly and is incorporated by reference herein. The sequence listing textfile “CLULA_014C3.txt” was created on Feb. 6, 2017, and is 14.5 bytes insize.

BACKGROUND OF THE INVENTION

Methods that allow highly specific detection of nucleic acids sequences,i.e., that permit discrimination between closely related sequences,including similar or related sequences differing by only a single base,are important in various applications, including, for example, detectingor distinguishing between multimember gene families, microRNAs (miRNAs),viral serotypes, or genetic variation between individuals. Highlyspecific detection methods are generally complex and require numeroussteps, and therefore, are not suitable for use in a high throughputformat. Methods that are amenable to high throughput, such as nucleicacid microarrays, typically rely solely on nucleic acid hybridizationfor specificity and therefore have a limited ability to distinguishbetween closely related sequences.

There is a need in the art for a method of detecting or discriminatingbetween nucleic acid sequences having high specificity and that isamenable to a high throughput format, such as microarrays, The methodwould be generally useful in a variety of applications requiring

SUMMARY OF THE INVENTION

In one aspect, the invention includes a method of preparing a detectablylabeled single-stranded polynucleotide target from a double stranded DNAcomprising the detectably labeled polynucleotide target hybridized to acomplementary polynucleotide. The double stranded DNA is contacted witha 5′ to 3′ exonuclease under suitable conditions to degrade at least aportion of the complementary polynucleotide to form a detectably labeledsingle-stranded polynucleotide target. The single-strandedpolynucleotide target includes one or more of: a cyanine dye moietypositioned between the second and third nucleotides from the 5′ end ofthe polynucleotide target; a cyanine dye moiety positioned between firstand second nucleotides from the 5′ end of the polynucleotide target anda modified linkage between the second and third nucleotides from the 5′end of the polynucleotide target; a cyanine dye moiety attached at the5′ end of the polynucleotide target, with the complementary strandmodified with a 5′-phosphate group or a primary amine with an aliphaticlinker arm connected to the polynucleotide via a phosphate linkage andthe first 5′-residue of said polynucleotide being an A or a T base; anda dye moiety attached at the 5′ end of the polynucleotide target and thefirst 5′-residue of said polynucleotide being a G or C base, with thecomplementary strand modified with a 5′-phosphate group or a primaryamine with an aliphatic linker arm connected to the polynucleotide via aphosphate linkage and the first 5′-residue of said polynucleotide beingan A or a T base.

In another aspect, the invention provides a method of generating adouble stranded DNA comprising a detectably labeled polynucleotidetarget hybridized to a complementary polynucleotide using primers thatinclude modifications that render the target and complementarypolynucleotides differentially sensitive to a 5′ to 3′ exonuclease.

Also provided is an array comprising a surface comprising epoxidemoieties and a plurality of oligonucleotides each comprising a 5′hydrazide linker attached to the surface of the array through a bondformed between the linker and an epoxide moiety, and methods of makingsuch arrays.

The invention further provides a buffer that can be used to make arrays.The buffer has a pH in the range of from about 4.0 to about 8.0 andincludes sodium phosphate (monobasic) in a concentration in the range offrom about 1 mM to about 1M, an ethylene oxide based nonionic detergentin a concentration in the range of from about 0.001% to about 1% (v/v),and ethylene glycol in a concentration in the range of from about 10% toabout 90% (v/v).

In yet another aspect, the invention provides a method of detecting thepresence of a specific nucleic acid sequence within a pool of detectablylabeled target polynucleotides by hybridization to one or moreoligonucleotide probes attached to a support. The support is contactedwith the polynucleotides under high stringency conditions in a buffercomprising a tertiary alkyl ammonium salt and formamide. Theunhybridized target polynucleotides are removed by washing the supportunder conditions similar to those used during the hybridization for aperiod about 30 minutes or less, the presence or absence of labeledtarget polynucleotides is detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an array system design wherein the target materialconsists of an antisense strand that is the product of a reversetranscription of an RNA sample, and is subsequently amplified throughasymmetric PCR or PCR followed by enzymatic digest of the sense strandprior to hybridization to immobilized sense-strand probe material.

FIG. 2 illustrates an array system design with the opposite target/probestrand orientation wherein the target material is the sense strand andthe probe material, which is immobilized on the microarray slidesurface, is the antisense strand.

FIG. 3 illustrates an array method that utilizes multiplexing throughthe use of sequence tags, employing an RNA virus model system as anexample.

FIG. 4 illustrates the use of the array invention in tandem with RNAlinkers.

FIG. 5 illustrates the effectiveness of the improved hybridizationbuffer and wash protocols (FIG. 5B) over conventional buffer and washprotocols (FIG. 5A) and the improved discrimination achieved using short“818” design oligonucleotide probes (FIG. 5B).

FIG. 6A shows differential migration of the individual single-strandedDNA model oligos, and differential exonuclease sensitivity of twofluorophores.

FIG. 6B illustrates sensitivity to exonuclease of DNA having various5′-end modifications.

FIG. 6C shows that the 5′-Cy3 modification confers resistance to lambdaexonuclease that is independent of the 5′-end base composition.

FIG. 6D shows T7 Exonuclease protection using multiple phosphorothioateinternucleoside linkages between 5′-end nucleotides.

FIG. 6E demonstrates T7 Exonuclease protection using one or lessphosphorothioate internucleoside linkage.

FIG. 7 demonstrates the improved sensitivity that is achieved by using asingle-stranded hybridization target compared to the same hybridizationtarget when it is double-stranded.

FIG. 8 illustrates that hybridization signals from the 5′-ILinkermodified oligonucleotide probes are significantly stronger than thehybridization signals from the same probes printed as amino-modified orunmodified oligonucleotides when hybridized to the same target under thesame conditions.

FIG. 9 compares different epoxide spotting solutions. Oligonucleotideprobes (41mers) were spotted at 40 uM concentration on an epoxide slide(Corning) using the Example 1 epoxide spotting buffer formulation (ESB),3×SSC, and three different commercially available spotting solutionsmarketed as either “specifically formulated for use on”, or “compatiblewith” the epoxide slide surface. The sub-arrays were then hybridizedwith complementary Cy3™-labeled oligonucleotide targets, washed, andscanned using a ScanArray® 5000 (Perkin-Elmer) at 62/62 laser(power/gain) settings.

FIG. 10 illustrates the effect of Nonidet P-40 as a component ofspotting buffer on probe spot size. Oligonucleotide probes (70mers) werespotted at 40 uM concentration on an epoxide slide (Coming) using theExample 1 epoxide spotting buffer formulation supplemented with varyingconcentrations of NP-40. The sub-arrays were then hybridized withCy3™-SpotQC, washed, and scanned, using the ScanArray® 5000(Perkin-Elmer) at 69/69 (optimal setting for the 50% GC probe) & 78/78(optimal setting for the 38.6% GC probe) laser (power/gain) settings.

FIG. 11 illustrates the results of Let7 model hybridizations using a“traditional” DNA microarray format using the method of the invention.

FIG. 12 illustrates the results of Let7 model hybridizations using a“reverse” DNA microarray format using the method of the invention.

FIG. 13 illustrates oligonucleotide probes (41mers) that were spotted at40 uM concentration on an epoxide slide (Corning) using differentspotting solution formulations that varied by source of sodium (300 mMsodium phosphate, monobasic vs. 3×SSC) and pH. The sub-arrays were thenhybridized with complementary Cy3™-labeled oligo targets and scanned,using the ScanArray® 5000 (Perkin-Elmer) at 60/60 laser (power/gain)settings.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel array system for nucleic acid sequencedetection having high specificity, capable of detecting geneticvariation and discriminating between variant sequences.

In one embodiment, the array comprises synthetic oligonucleotide probesspotted on a solid surface that can be hybridized to a single-strandedtarget that is modified to permit detection. The labeled target can beprepared by PCR amplification in which one of the primers modified at ornear the 5′-end to prevent degradation by a 5′- to 3′-exonuclease,thereby allowing preferential exonuclease degradation of the unprotectedcomplementary strand, thus producing a final product comprisingsingle-stranded target from a double-stranded amplification product. Theinvention is also intended to encompass applications in which the finalproduct formed is enriched with respect to the single-stranded target,although some of the complementary strand may remain.

The invention may include various aspects that enhance sensitivity orspecificity of detection of target oligonucleotides, or reduce processtime, or enhance throughput, which may be used alone or in variouscombinations. Aspects of the invention include probe design, probeattachment, probe spotting buffer composition, preparation of labeledsingle-stranded targets, hybridization buffer, and hybridization andwash conditions.

Probe Design.

Generally, shorter probes have a high specificity but a low sensitivitywith a greater chance of cross-hybridization. The exception would be avery short sequence that could occur multiple times in a genome,therefore being less specific (e.g., a 6-base sequence occurs on averageonce every 4000 bases. Historically, microarrays have employed probesof >20 bases to improve sensitivity. Because all nucleic acidhybridization events on a microarray occur simultaneously underidentical conditions, traditional methods use probes designed tonormalize the Tm. In other words, probes are designed such that themelting temperature of the each probe is substantially the same, so thatall probes can be hybridized in parallel in the same buffer at the sametemperature. DNA base composition varies widely within localized regionsof a genome. The location of a SNP defines the sequence context of aprobe to detect that SNP, and the local GC content of that sequence canvary widely. As a result, Tm normalized probes will vary significantlyin length. For example, keeping Tm fixed at 70° C., probe length couldvary from 14 bases to >50 bases, depending on relative GC content. Whenusing this approach, short probes will show superior mismatchdiscrimination compared to long probes. Long probes, however, cannot beshortened without lowering Tm, and lower Tm probes cannot be hybridizedsimultaneously with other probe sequences having a higher Tm. Affinitycan be increased using modified bases, such as locked nucleic acids(LNAS), however these modifying groups are expensive and their use isunrealistic for high content arrays.

In the present invention, probe sequences are short (<20 bases) and allprobes have the same, or substantially the same, length. Bysubstantially the same length, it is meant that at least 90% of theprobes in the array vary by no more than plus or minus two bases fromthe median probe length. Suitably, at least 90% of the probes fallwithin plus or minus one base of the median probe length. In oneembodiment, the probe is 17 nucleotides in length.

Tm normalization is achieved by employing hybridization buffers thatreduce the affinity differences between GC and AT base pairs, thusreducing sequence dependence of the Tm. Hybridization buffers andconditions according to the present invention are described below.

In a further embodiment, when the probe is used for SNP detection, thebase corresponding to the SNP is positioned centrally within the probesequence. In the case of a 17mer probe, there are 8 bases 5′- and 8bases 3′- to the centrally located SNP base. This design will bereferred to as an “818” design hereafter. If more than one polymorphismis present, the variable bases may be positioned as near to the centerof the probe as possible. In another embodiment, probe length can bevaried, and probes of 15-20 bases length can be used. In general, probesunder 15 base length will have reduced sensitivity and probes greaterthan 20 base length will have reduced specificity.

Attachment Chemistry.

In another aspect, arrays are formed by applying probes to a solidsurface so as to enhance signal intensity following hybridization.Sensitivity of detection is enhanced by improving attachment of theoligonucleotide to the solid surface. In one embodiment, ahydrazide-modified probe is attached to an epoxide surface. Thehydrazide improves attachment of the oligo probe to the solid surface.For short 17 base probes, the use of a hydrazide attachment to epoxidesurfaces enhances signal intensity by greater than 3-fold. Thisattachment can be used for probes of any length, but is particularlyhelpful with shorter probes, because as oligo length decreases, thenumber of available nucleo-base amine groups available for attachment ofthe probe to epoxide also decreases. Thus, hydrazide attachmentchemistry is especially helpful when using shorter probes.

Amino modified oligonucleotides react with active epoxide slides,opening the epoxide ring and forming a stable amide bond. Hydrazidemodified oligonucleotides react in the same manner, but the attachmentis stronger because hydrazide is a stronger nucleophile. The reactionalso occurs at a lower pH whereas amines are protonated at low toneutral pH conditions. As demonstrated in Example 12, the hydrazidemodified oligonucleotides demonstrate a more intense signal compared tounmodified oligonucleotides and amino-modified oligonucleotides,especially at lower pH conditions. Preferably, the pH is less than 8.5.Suitably, the pH is in the range of from about 4 to about 8.5. Morepreferably, the pH is in the range of from about 4.5 to 5.5.

Spotting Buffer.

A spotting buffer formulation according to the present invention canfurther increase signal intensity and improve spot morphology. Oneaspect of the present invention is a spotting buffer that improves thesignal intensity and spot morphology of oligonucleotides printed onepoxide surfaces, and is suitable for use in conjunction with hydrazideattachment chemistry of the invention. The epoxide spotting buffer (ESB)of the present invention comprises a monobasic sodium phosphate (lowpH), an ethylene oxide based non-ionic detergent such as Nonidet P-40(NP-40), and ethylene glycol. The ESB can be used to more efficientlyattach either unmodified or amino-modified oligo probes through acovalent linkage to an epoxide-surface microarray slide. The ESB alsoenables the immobilization of an oligo probe to the epoxide surfacethrough a covalent linkage utilizing a hydrazide-modifiedoligonucleotide, allowing efficient immobilization of very shortoligonucleotide probes (16-17mers). In addition, ESB can be made-up as aconcentrated formulation (e.g., a 2× formulation) that can be used to“rescue” oligo probes that have been previously resuspended in a varietyof different spotting solutions.

Nonidet P-40 (NP-40), which is currently available under the name IgepalCA-630, is an ethylene oxide based non-ionic detergent, which is morecompatible with epoxide surfaces than are other classes of detergents.It is also compatible with the anti-evaporation additive ethyleneglycol. It is expected that other ethylene oxide based non-ionicdetergents such as Triton X-100, an ethylene oxide based non-ionicdetergent, but differs from NP-40 in the number of ethylene oxide units,could also be used in the EBS of the present invention.

The use of monobasic sodium phosphate in the composition of an epoxidespotting solution helps to attain maximal attachment of the 5′ hydrazidemodified oligonucleotide probes to an epoxide surface. Increasing the pHof the spotting solution by either titrating in sodium hydroxide (NaOH)or by changing the source of sodium (3×SSC) results in decreasedhybridization signal, suggesting decreased oligonucleotide probeattachment density within the probe spot and also increases probe spotsize (see FIG. 9). The factors showing the greatest impact on probeattachment density seems to be pH, with slightly acidic pH beingsuperior, and use of the 5′ hydrazide modifier instead of anamino-modifier or no modification. The spotting buffer components thathave the greatest impact on spot size seems to be the source of sodiumand the detergent, with monobasic sodium phosphate and NP-40 beingpreferred.

The composition of the final 1× spotting solution is compatible withformulation as a 2× concentrate (all components remain in solution).This, in turn, allows a 1:1 dilution with oligo probe material that hasalready been resuspended in a different, sub-optimal spotting solution,effectively rescuing the probe material for efficient immobilization onepoxide microarray slides. For example, large sets of oligonucleotideprobes may exist in a laboratory which were previously suspended inwater, 3×SSC, or other sub-optimal spotting solution. Use of a 2×concentrate of the new ESB can improve spotting of these probes withoutrequiring complete buffer substitution, which may not be feasible forlow concentration, low yield probe sets.

Preparation of Labeled Targets.

The probes on a spotted microarray are hybridized to a target nucleicacid that is labeled to facilitate detection. In general, targetsequences are seldom present in biological samples in sufficient amountsto permit direct detection. It is therefore envisioned that someamplification process will be performed on a heterogeneous biologicalnucleic acid sample and that the amplified product will be hybridized tothe array. PCR based methods resulting in exponential targetamplification are powerful. However, the products of exponential PCR aredouble-stranded, with each strand being present in equimolar amounts.With denaturation and subsequent hybridization, the complementarystrands of the amplified targets can re-hybridize to each other andcompete with probe hybridization. If the target nucleic acid is long,the stability of the target-target duplex will be greater than thetarget-probe duplex and this reaction will be favored, resulting in aweak or absent hybridization signal on the array. It is thereforepreferable to use single-stranded targets such that the sole or primaryspecies is the strand complementary to the immobilized probe sequence.

Many different amplification methods exist which can producesingle-stranded labeled targets. For example, linear amplification canbe performed using thermal cycling (similar to cycle sequencing). Whilethis method produces single-stranded targets, the amplification power islow and typical yields are 30-100 fold amplification. PCR based methodscan give 10⁸ fold amplification. Asymmetric PCR can be used which startsas exponential PCR and then shifts to linear amplification afterconsumption of a limiting primer. Although more powerful than simplelinear amplification this method still produced far less amplificationthan true exponential PCR. As one aspect of the present invention, animproved method of generating single-stranded labeled template isdescribed that employs full exponential PCR followed by selectivedegradation of the undesired target strand.

The array system and its methods of use include a method for identifyingor typing genetic strains by amplifying nucleic acid molecules (DNA orRNA) of a sample with one or more primers that are specific to aconserved region of a genetic strain being assessed (e.g., PCR,asymmetric PCR, RT, or RT followed by either PCR or asymmetric PCR), tothereby obtain an amplified nucleic acid product. The methods alsoinvolve contacting the amplified nucleic acid product with one or moregenetic strain specific probes having a nucleic acid sequence that isspecific for only one genetic variant in a group being assessed, whereinthe nucleic acid sequence includes between about 9 and 25 nucleic acidbases. The presence of one or more hybridization complexes with agenetic strain specific probe indicates the presence of one or morespecific genetic strain, and the absence of one or more hybridizationcomplexes with a genetic strain specific probe indicates the absence ofthe specific genetic variant in the sample. Amplification of the nucleicacid molecules can be obtained using PCR, asymmetric PCR, RT, or RTfollowed by either PCR or asymmetric PCR.

The target that is amplified can originate from any genetic sample,including samples from the feces, saliva, sputum, aspirate, blood,plasma, cerebrospinal fluid, aspirate, tissue, skin, urine, mucus, etc.The methods of this invention can work with a sample containing a vastamount of non-specific genetic material, which is not related to thegenetic material of interest.

When amplification occurs via PCR, asymmetric PCR, RT, or RT followed byeither PCR or asymmetric PCR, select primers containing one or twophosphorothioate linkages can be utilized (PS-primer). Preferably twolinkages are PS modified. The resulting nucleic acid strands generatedfrom the PS-primers are protected from degradation by a 5′-to-3′exonuclease that will degrade the complementary strands generated by theunmodified (non-PS) primer, thereby creating a single-stranded targetthat is suitable for use in the array system. The PS-primer can bemodified with a fluorophore at the 5′ end, said fluorophore can beoptionally attached via a phosphorothioate linkage but is not required.Ideally the fluorophore will be suitable for direct detection bymicroarray scanning fluorometers. Examples include Cy3 and Cy5 dyes.Other dyes can be used. In one embodiment, two phosphorothioate bondsare positioned in the internucleoside linkages between the first andsecond and second and third bases from the 5′-end of the primeroligonucleotide. This configuration provides substantially completeprotection from degradation using the enzyme T7 Exonuclease. If a5′-fluorophore is present, the linkage between the fluorophore and thefirst nucleotide at the 5′ end can employ a phosphodiester bond. It isnot necessary to modify this bond; however use of PS bond between thefluorophore and the first base does not impair function and can be used.In addition, there is partial protection when only a single PS bond ispresent between the first and second nucleotide positions in conjunctionwith the presence of a 5′-fluorophore; no partial protection is evidentwhen a single PS bond between the first and second nucleotide positionsis present in the absence of the 5′-fluorophore. However, a single PSbond placed between the second and third nucleotide positions conferssubstantially complete protection from T7 Exonuclease digestion if it ispresent in the absence of a 5′-fluorophore. This is consistent with T7Exonuclease cleaving initially in a dinucleotide unit and thenproceeding by cleaving mononucleotides until it dissociates. The methodsof target preparation taught in the present invention enable use of fullexponential PCR, produces single-stranded target molecules, and employsprimers with the minimum modification necessary to protect the desiredtarget strand from exonuclease degradation. Utility and use ofsingle-stranded targets prepared using the method of the invention isshown in Example 3, FIGS. 7 and 8.

Another embodiment of the invention employs an internally placedfluorophore located near the 5′-end of the primer. If a Cy3 or othercyanine dye is positioned between the second and third bases from the5′-end of the primer oligonucleotide, this strand of the resulting PCRproduct is resistant to degradation by T7 Exonuclease, even in theabsence of phosphorothioate or other nuclease-resistant modifications.Internal placement of the cyanine dye between the first and second basesfrom the 5′-end of the primer oligonucleotide provides for partialnuclease resistance; in this case, incorporation of a singlephosphorothioate bond or other nuclease-resistant modification betweenbases 2 and 3 from the 5′-end will, together with the internal cyaninedye, protect that strand of the resulting PCR product from degradationby T7 Exonuclease. The methods that allows T7 Exonuclease degradation ofa complementary strand to produce single-stranded targets is describedin Example 2, FIGS. 6D and 6B.

The methods above employ the enzyme T7 Exonuclease to convert adetectably labeled double-stranded nucleic acid into a detectablylabeled single-stranded nucleic acid suitable for hybridization to shortprobes oligonucleotides. In yet another embodiment of the invention, amethod to produce detectably labeled single-stranded nucleic acidspecies using Lambda Exonuclease is described. Lambda Exonuclease is aprocessive 5′ to 3′ nuclease that removes 5; mononucleotides fromduplexed DNA. Initiation of degradation is typically thought to requirea phosphate group at the 5′-end. of the degraded strand. The presentinvention includes novel methods to trigger strand degradation by LambdaExonuclease which do not require a 5′-phosphate. The presence of avariety of non-phosphate modifying groups at the 5′-end of the nucleicacid will trigger degradation of that strand of a double-strandednucleic acid by Lambda Exonuclease. A 5′-amino-modifier (primary aminelinked to the nucleic acid by a 6 carbon alkyl linker and a phosphatebond) will trigger Lambda Exonuclease attack. Cyanine dyes, such as Cy3or Cy5, placed at the 5′-end of one strand of a double-stranded nucleicacid are resistant to Lambda Exonuclease degradation. Use of twodifferentially modified oligonucleotide primers, one having a 5′-cyaninedye and the second having a 5′-amino-modifier, can be employed togenerate a double-stranded PCR product having a 5′-cyanine dye on onestrand and a 5′-amino-modifier on the complementary strand. Thisdouble-stranded nucleic acid will be a substrate for Lambda Exonucleasedegradation such that the amino-modified strand will be substantiallydegraded while the cyanine dye labeled strand will remain intact,resulting in a detectably labeled single-stranded nucleic acid that canbe employed in hybridization experiments.

Additional 5′-modifying groups can be used to trigger Lambda Exonucleaseattack. While cyanine dyes do not trigger degradation, otherfluorophores will trigger attack by Lambda Exonuclease. For example,fluorescein will trigger degradation, but only if the first 5′ base ofthe nucleic acid is an A or a T residue. A 5′-fluorescein group adjacentto a C or G residue will be resistant to attack by Lambda Exonuclease.It will be clear to one skilled in the art that the differentialreactivity of cyanine and fluorescein dyes can be used to make adual-labeled double-stranded nucleic acid, one strand having a5′-cyanine dye and the complementary strand having a 5′-fluorescein dye,and that this double-stranded nucleic acid will be reduced to asingle-stranded cyanine-dye labeled species if a 5′-A or 5′-T residue isadjacent to the fluorescein modifier. This strategy permits control ofdegradation based upon sequence context.

Hybridization Buffer.

Use of short oligonucleotide probes of identical length in a traditionalsodium based hybridization buffer will result in widely variablehybridization results dependant upon the Tm of each probe. One aspect ofthe invention is use of hybridization buffer that minimizes the Tmdifference between oligonucleotides of different sequence. In the buffersystem described herein, hybridization is regulated more by the lengthof perfect base pairs present between probe and target such that theeffects of mismatches are magnified while variations in sequence whichdo not contribute to mismatch are minimized. In one embodiment, thehybridization buffer is a combination of Tris at a pH around 8; EDTA;Sarkosyl; Ovalbumin; CTAB; Ficoll Type 400; PVP-360; tetramethylammonium chloride (TMAC); formamide; and Cot-1 DNA. In anotherembodiment, the composition of the hybridization buffer is: 37.5 mM TrispH 8, 3 mM EDTA, 0.25% Sarkosyl, 0.4 mg/mL Ovalbumin, 1 mM CTAB, O.4mg/mL Ficoll Type 400, O.4 mg/mL PVP-360, 2.5M TMAC, 10% Fonnamide, 10ug/mL Cot-1 DNA. The composition of the 1×SNP Wash Buffer 1 is: 2.5MTMAC, 0.2% Sarkosyl.

Typically, microarray hybridization is done under relativelynon-stringent conditions to maximize hybridization and thereby maximizesignal. The hybridization step is usually long, typically overnight (>12hours). This permits imperfect hybridization events to take place,decreasing specificity. Specificity is then improved by using stringentwash conditions. The method of the present invention has reversed thisapproach and obtains greater mismatch discrimination in shorterhybridization periods. Unlike buffers described in the prior art (seeU.S. Pat. No. 6,361,940), the advantage of the proposed system is thatit analyzes the probe/target interaction at the hybridization (or ON)step. Post hybridization wash steps are rapid and are less stringentthan the original hybridization conditions. Most hybridizations methodsuse wash conditions that are more stringent than the hybridizationconditions and therefore analyze samples that have been hybridized andthen stripped off. Traditional approaches employ the concept thatmismatches can be preferentially stripped while leaving theperfect-matched hybridization duplexes intact. The conditions describedherein achieve maximal signal at 2.25 hours hybridization at 50° C.,then a 15 minute wash at 50° C. in 1×SNP Wash buffer 1, followed by a 1minute rinse in 2×SSC at room temperature, followed by a brief rinse(1-2 seconds) in 0.2×SSC at room temperature, spin dry the slide, andthen scan to visualize.

Traditional array hybridization protocols utilize a stringent wash (thearray is washed with a low-salt buffer at a constant temperature) toremove non-specific pairs that have also hybridized. There is often afirst wash that is moderately stringent followed by one or more higherstringency washes that preferentially remove the non-specifichybridization targets. However, specific and non-specific hybridizationtargets are removed resulting in less signal while maintaining the samedifferential hybridization.

The proposed hybridization and wash methods provide hybridizationconditions that enhance selective hybridization of the specific target.A non-stringent wash step exploits the variable of target concentrationin the hybridization kinetics equation; i.e., lower the concentration ofthe target and the non-specific hybridization events are selectedagainst while the maintained salt concentration (or stringency) helps tostabilize (or maintain) the hybridization of the specific target in thelowered target concentration environment. Both improved specificity andimproved sensitivity (increased signal) result through use of these newmethods. Demonstration of the utility of the new hybridization buffer,wash buffer compositions and hybridization and wash protocols is shownin Example 1, FIG. 5.

Hybridizations using the new hybridization buffer can be conducted forexample, at 50° C. for from 1-4 hours, of similar with hybridizations of2-2.5 hours being preferred. Washes were performed in a buffer having anionic strength similar to that of the hybridization buffer, (2.5M TMAC,0.2% Sarkosyl) and without formamide at 50° C. for 15 minutes, withwashes of 10-30 also giving good results.

One suitable hybridization buffer includes 37.5 mM Tris pH 8, 3 mM EDTA,0.25% Sarkosyl, 0.4 mg/mL Ovalbumin, 1 mM CTAB, 0.4 mg/mL Ficoll Type400, 0.4 mg/mL PVP-360, 2.5M TMAC, 10% Formamide, 10 ug/mL Cot-1 DNA).However, as one of skill in the art will appreciate, minor variations tothis formulation can be made without affecting its performance and areintended to fall within the scope of the invention. For example, thebuffer may include a higher or lower concentration of Tris buffer at apH range of from 7-8.5, from 1-10 mM EDTA, from 0.1 to 1% Sarkosyl from0.1-1 mg/ml ovalbumin, from 0.1-5 mM CTAB, from 0.1-1.0 mg/mL FicollType 400, 00.1-1.0 mg/mL PVP-360, from 2.0-3.0M tetramethyl ammoniumchloride (TMAC), from 0.20% Formamide, and from 1-100 ug/mL Cot-1 DNA.

General categories of applications in which the methods of the inventionis useful include, but are not limited to, genome-wide SNP analysis (oreven subsets of the genome SNP set), genetic strain typing, and miRNAsequence family detection and discrimination. The specific strategyemployed for the target generation will most likely change depending onthe specific application. All three applications will have the basicrequirement that a sequence tag (or tags) would be introduced to allowfor amplification using either a simple primer pair (or a limited primerpool) such that a specific strand can be labeled and protected from thedigestion step (such as T7 exonuclease digestion). FIGS. 1-4 illustrateexamples of the multiple labeling strategies that could be employed,depending on the specific application (or embodiment). The geneticstrain typing application would use a primer set that is containedwithin a sequence common to all strains while being unique to thespecific genetic organism (see FIGS. 1 & 2). This would generatesuitable target material while limiting the amplification ofnon-specific genetic material. FIG. 3 illustrates how universal-tagsequences could be implemented in an embodiment for multiplexed analysisof genetic strains from different genetic organisms. An embodiment fordiscriminating the variation between closely related miRNAs, would alsouse a similar “universal sequence tagging” method (see FIG. 4). Here the“universal tagging sequences” would be introduced via a 3′ and 5′ linkerand the “universal primers” would be their complements. Linkers of thiskind are already employed for miRNA cloning and can be directly adaptedto this new application (see U.S. patent application 60/946,922, whichis incorporated by reference).

In one embodiment, the invention pertains to the use of linkers used toattach specific and unique sequences to the 3′-end of small RNAs,such-as miRNAs, which can be subsequently used as a primer site forreverse transcription (RT) followed by immobilization of the RT productonto a microarray slide. The 3′-linker can be adapted from designsdescribed by Bartel (Science 2001; 294:858-62), which is incorporated byreference, and the immobilization of the RT product onto a microarrayslide as described by Rogler et al. (Nucleic Acids Res., 2004; 32:e120),which is incorporated by reference.

The current favored method for placing a suitable nucleic acid sequenceat the 3′-end of small RNAs for use as a reverse transcription primerbinding site is poly-A tailing. This method utilizes the RNA poly-Apolymerase (PAP) enzyme from yeast or E. coli to add a sufficient numberof adenosine ribonucleotides onto the 3′-end of the small, non-polyA-containing, RNAs to allow the use of oligo-dT as a reversetranscription primer (Martin and Keller, RNA, 4, 226-230 (1998); Fu etal., 2006). The poly-A tailing method was developed to overcome theshortcomings of using random hexamer priming for reverse transcriptionon short RNA templates (and other non-poly adenylated RNAs). Otherspecific methods exist whereby the polynucleotide tailing adds a numberof other mononucleotides so that an RT primer different than poly-A canbe utilized (Cho 2007; Kwak and Wickens, RNA (2007), 13:860-867;Rissland, Mol. Cell Biology, 27: 3612-3624 (2007)).

In one embodiment, the 3′ linker is attached to the 3′-end of the smallRNA sample, via a T4 RNA ligation reaction, followed by an RT reactionusing as primer a 5′-hydrazide modified oligonucleotide that iscomplementary to the 3′-linker sequence. Then the RT product is attached(spotted) onto a microarray glass slide that would be suitable forhybridization with a fluorophore-labeled synthetic oligonucleotideprobe. In another embodiment, this approach could be reversed where theRT primer is modified with a 5′-fluorophore, instead of a 5′-hydrazide,and the resulting product functions instead as labeled target, which canbe hybridized to a microarray containing immobilized synthetic oligoprobes (“traditional” array format).

The embodiment also provides a method to incorporate the addition of a5′ linker after the 3′-linkering step and before the RT step to providea unique 5′-sequence tag that would be useful for dual-labeled PCRassays of the same material immobilized on the microarray glass slide(see FIG. 4), or for a general amplification method of low (or limited)quantities of starting material.

This method is more efficient and potentially a more sensitive method ofgenerating suitable material for reverse transcription from a small RNApool fraction of a total RNA isolation. The method could also be usedfor the integration of multiple assays (microarray and dual-labeled PCR)of the same RNA material from a sample of interest (i.e.; parallel assayformats on both the mRNA and “small” RNAS fractions from the same RNAisolation sample of interest). Universal PCR strategies employingsequence-tagged primers are known in the art (see Lao et al 2007 (U.S.Pat. No. 7,176,002)).

The term “microarray” is used interchangeably with “array, gene chip,”“DNA chip,” “biochip,” and refers to a plurality of spots ofoligonucleotides on a solid support for use in probing a biologicalsample to determine gene expression, marker pattern or nucleotidesequence. Examples of supports include, but are not limited to, glass,silica chips, nylon (polyamide) membrane, polymer, plastic, ceramic,metal, coated on optical fibers, or infused into a gel matrix.

The proposed methods could also be used in liquid array platforms, suchas with polystyrene beads, or with acid-etched bar-coded fiber opticcable (see CyVera), gold nanoparticles, transponders, or silicon-basedbeads. The methods could also be utilized in in situ synthesis arrayplatforms (see Affymetrix and Nimblegen).

The solid support can also be coated to facilitate attachment of theoligonucleotides to the surface of the solid support. Any of a varietyof methods known in the art may be used to immobilize oligonucleotidesto a solid support. The oligonucleotides can be attached directly to thesolid supports by epoxide/amine coupling chemistry. See Eggers et al.Advances in DNA Sequencing Technology, SPIE conference proceedings(1993). Another commonly used method include the non-covalent coating ofthe solid support with avidin or streptavidin and the immobilization ofbiotinylated oligonucleotide probes.

In one embodiment, amplification includes or is optionally followed byadditional steps, such as labeling, sequencing, purification, isolation,hybridization, size resolution, expression, detecting and/or cloning.

In one embodiment, after a round of RT-PCR with a single pair of primersof low degeneracy, the RT-PCR product is labeled using an anti-senseprimer (e.g., with Cy-3) during amplification. Single stranded targetDNA is obtained by enzymatic degradation of the unlabeled sense standfollowed by column purification of the labeled antisense strand. Singlestranded antisense target DNA can also be obtained by asymmetric PCR,described herein, using excess labeled sense primer. Conversely, theRT-PCR product can be labeled with a sense primer when the probe is inthe antisense conformation.

Several labels exist to facilitate detection of a nucleic acid moleculecomplex. Techniques for labeling and labels, that are known in the artor developed in the future, can be used. In a preferred embodiment, thelabel is simultaneously incorporated during the amplification step inthe preparation of the sample [nucleic acids. For example, PCR withlabeled primers or labeled nucleotides will provide a labeledamplification product. The nucleic acid (e.g., DNA) is amplified in thepresence of labeled deoxynucleotide triphosphates (dNTPs). In apreferred embodiment, transcription amplification, as described above,using a labeled nucleotide (e.g., fluorescein-labeled UTP and/or CTP)incorporates a label into the transcribed nucleic acids.

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. The mostfrequently used labels are fluorochromes like Cy3, Cy5 and Cy7 suitablefor analyzing an array by using commercially available array scanners(e.g., Axon, General Scanning, Genetic Microsystem, and Perkin Elmer).Other labels that can be used in the present invention include biotinfor staining with labeled streptavidin conjugate, magnetic beads (e.g.,Dynabeads™), dendrimers, fluorescent proteins and dyes (e.g.,fluorescein, Texas red, rhodamine, green fluorescent protein, and thelike, see, e.g., Molecular Probes, Eugene, Oreg., USA), radioactivelabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radishperoxidase, alkaline phosphatase and others commonly used in an ELISA),and colorimetric labels such as colloidal gold (e.g., gold particles inthe 40-80 nm diameter size range scatter green light with highefficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include WO 97/27317, and U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

A fluorescent label provides a very strong signal with low background.It is also optically detectable at high resolution and sensitivitythrough a quick scanning procedure. The nucleic acid samples can all belabeled with a single label, e.g., a single fluorescent label.Alternatively, in another embodiment, different nucleic acid samples canbe simultaneously hybridized where each nucleic acid sample has adifferent label. For instance, one target could have a green fluorescentlabel and a second target could have a red fluorescent label. Thescanning step will distinguish cites of binding of the red label fromthose binding the green fluorescent label. Each nucleic acid sample(target nucleic acid) can be analyzed independently from one another.

The sample can be purified to reduce the overall slide background (i.e.;the inter-spot space area) that would be caused by the “un-used”fluorophore-labeled primer. This in-turn would impact the overallsensitivity of the array, affecting the signal-to-noise ratio. It wouldnot have any effect on the selectivity of the array probe designs (i.e.;the cross-hybridization signal). Once the sample is prepared, it can besubjected to the nucleic acid molecules of the present invention forhybridization. Hybridization refers to the association of single strandsof oligonucleotides through their specific base-pairing properties toform a complementary double-stranded molecule. With respect to thepresent invention, the labeled DNA of the sample hybridizes with theoligonucleotides on the solid support. Hybridization conditions includevariables such ‘as temperature, time, humidity, buffers and reagentsadded, salt concentration and washing reagents. Preferably,hybridization occurs at high stringency conditions and examples ofsuitable stringency conditions are described herein. Methods forhybridization are known, and such methods are provided in U.S. Pat. No.5,837,490, by Jacobs et al. The solid support can then be washed one ormore times with buffers to remove unhybridized nucleic acid molecules.Hybridization forms a complex between the nucleic acid of the presentinvention and nucleic acid of the sample.

The methods of the present invention also involve determining the levelor percentage of a particular genetic variant type in a sample (such asthe expression levels of the various Let7 miRNA family members in aparticular sample). Data can be generated for mean detection levels orpercentage of known quantities of a genetic variant type and can be usedto compare a sample of unknown quantity to determine the level orpercentage of the genetic variant type in the sample. In one embodiment,threshold levels or percentages (e.g., low, medium and high) of geneticvariant types can be established using known quantities of the variants,and compared to an unknown level or percentages of variants in a sample.Detection of one or more variant above the high threshold levelsignifies high quantities of the particular variant, detection of amedium threshold level indicates a mid-level quantity of the variant inthe sample, and detection-of variants below the low threshold levelsindicate low quantities of the variant in the sample.

The present invention includes methods of making an array. The methodincludes selecting a solid support, as described herein. In oneembodiment, epoxide slides are used. In particular, arrays can besynthesized on a solid substrate by a variety of methods, including, butnot limited to, light-directed chemical coupling, and mechanicallydirected coupling. See Pirrung et al., U.S. Pat. No. 5,143,854 (see alsoPCT Application No. WO 90/15070) and Fodor et al., PCT Publication Nos.WO 92/10092 and WO 93/09668 which disclose methods of forming vastarrays. See also, Fodor et al., Science, 251, 767-77 (1991). One exampleof synthesizing a polymer array includes the VLSIPSTM approach.Additionally, methods which can be used to generate an array ofoligonucleotides on a single substrate can be used. For example,reagents are delivered to the substrate by either (1) flowing within achannel defined on predefined regions or (2) “spotting” on predefinedregions. However, other approaches, as well as combinations of spottingand flowing, or other approaches can be employed.

The method further includes preparing the nucleic acid molecules forattachment to the solid support. Optionally, a spacer that provides aspace between the support and the capture nucleotide sequences can beused to increase sensitivity of the array. A spacer that can be usedwith the present invention includes any molecular group that allows thenucleic acid molecule to remain off of or separated from the support.Another example of a spacer is a hexaethylene glycol derivative for thebinding of small oligonucleotides upon a membrane. Patent publicationNo.: EP-0511559. In one embodiment of the invention, the nucleic acidprobes of this invention comprise at least two parts, the specificprobe, and the spacer/linker section. The specific probe portioncomprises about 9-30 nucleic acids or nucleic acid mimetics (e.g.,PNAs). The spacer/linker is comprised of anything that positions thespecific probe away from the substrate and that adheres or attaches thespecific probe to the substrate. Alternatively, probes can be attachedto a gel, in which case, a spacer/linker is not necessary.

The nucleic acid molecules of the present invention can also be preparedto promote attachment to the solid support chosen, or to react with acoating placed on the support. The solid support can be coated topromote adherence to the support, and once the nucleic acid molecule isapplied, in some cases ultraviolet irradiation allows for DNA fixation.For example, the nucleic acid molecules of the present invention or thesolid support can be modified to react with substrates including aminegroups, aldehydes or epoxides to promote attachment. Methods, now knownor developed later, for promoting attachment of the nucleic acid to thesolid support can be used.

The present invention includes kits. Kits can include the array of thepresent invention, as described herein. Kits can also include reagentsthat are used to carry out hybridization. Examples of such regentsinclude labeling reagents, primers that are specific to a conservedregion of the serotypes being assessed (labeled and/or unlabeled),buffers and washing solutions. Labeling reagents include labels, asdescribed. herein (e.g., fluorescent dyes, streptavidin conjugate,magnetic beads, dendrimers, radiolabels, enzymes, colorimetric labels,nanoparticles, and/or nanocrystals) including cyanine dyes such as Cy3and Cy5. The kit can also include software use to analyze the results,as described herein.

As used herein, the terms “DNA molecule” or “nucleic acid molecule”include both sense and anti-sense strands, cDNA, complementary DNA,recombinant DNA, RNA, wholly or partially synthesized nucleic acidmolecules, PNA and other synthetic DNA homologs. A nucleotide “variant”is a sequence that differs from the recited nucleotide sequence inhaving one or more nucleotide deletions, substitutions or

As used herein, an “isolated” gene or nucleotide sequence which is notflanked by nucleotide sequences which normally (e.g., in nature) flankthe gene or nucleotide sequence (e.g., as in genomic sequences). Thus,an isolated gene or nucleotide sequence can include a nucleotidesequence which is designed, synthesized chemically or by recombinantmeans.

Also encompassed by the present invention are nucleic acid sequences,DNA or RNA, PNA or other DNA analogues, which are substantiallycomplementary to the DNA sequences and which specifically hybridize withtheir DNA sequences under conditions of stringency known to those ofskill in the art. As defined herein, substantially complementary meansthat the nucleic acid need not reflect the exact sequence of thesequences of the present invention, but must be sufficiently similar insequence to permit hybridization with nucleic acid sequence of thepresent invention under high stringency conditions. For example,non-complementary bases can be interspersed in a nucleotide sequence, orthe sequences can be longer or shorter than the nucleic acid sequence ofthe present invention, provided that the sequence has a sufficientnumber of bases complementary to the DNA of the serotype to beidentified to allow hybridization therewith.

The following non-limiting examples are intended to be purelyillustrative, and should not be construed as in any way limiting itsscope.

Example 1 Probe Design, Attachment Chemistry & Hybridization Buffer

The following example describes attachment of oligonucleotides tosurfaces, including epoxide surfaces, hydrazide attachment chemistry(and the increase sensitivity it affords), the benefit of use of shortnucleic acids probes to improve specificity (specifically a 17mer havingan “818” probe design), and use of new hybridization and wash conditionsand protocols to enhance detection and specificity of nucleic acidsequence detection in an array or microarray assay format.

Probes for several single nucleotide polymorphism (SNP) sites within thecotton genome were designed using thermodynamic algorithms to normalizemelting temperatures (Tm) of perfect-match hybridization events whilesimultaneously maximizing the predicted Tm differences for allelicmismatches. Due to sequence content variation in SNP sites, Tmnormalized probes had lengths that varied from 17 to 29 bases, as wellas having variable SNP locations within each probe design, although eachSNP was more or less centralized within the probe's design. An exampleof this can be seen below with the “Tm Balanced” probe set.Oligonucleotide probes were synthesized by Integrated DNA Technologiesand provided as unpurified, desalted preparations having either a 5′-OH(unmodified) or a 5′-ILinker (or hydrazide) modification consisting ofthe following moiety.

For one probe, two sites of sequence variation or SNPs were presentwithin the selected sequence, representing “linked” SNPs that arenaturally encountered in the cotton genome.

Short 17mer probes were also designed at each SNP site, withoutconsideration for Tm. Instead, the SNP was simply positioned centrallywithin the probe sequence with 8 bases of sequence 5′ to the SNP siteand 8 bases of sequence 3′ to the SNP site. This collection is referredto as the “818” probe set. Unmodified and 5′ILinker modifiedoligonucleotides were also synthesized for this set by Integrated DNATechnologies and provided as unpurified, desalted preparations.

The sequences of both probes sets are shown below, with “Len” indicatinglength in bases, and “PM”, “MM”, and ΔTm denoting the predicted Tm (indegrees Celsius) of the perfect match hybridization (PM), the Tm of themismatch allele hybridization event (MM), and the calculated predicteddifferential (ΔTm) between the Tm's of the perfect match and mismatchhybridization, respectively. The program “HyTher” was employed tocalculate Tm of perfect match and mismatch pairs, using settings are 50mM monovalent; 0 molar Mg²⁺; 37 degrees C. hyb; and strandconcentrations of 200 nM.

Tm Balanced Probe Set: Name Len PM MM ΔTm SEQ ID NO: 1 TAACACC GCCAATGTCACA Tm SNP 5-A 19 54.3 47.4  6.9 SEQ ID NO: 2 CTAACACC ACCAATGTCAGAAG Tm SNP 5-D 22 53.9 46.9  7.0 SEQ ID NO: 3 TTAAAAA GGCGATACC G GGG Tm SNP 9-A 20 54.9 41.9 13.0 SEQ ID NO: 4 GAATTAAAAA TGCGATACC A GGGA Tm SNP 9-D 24 53.8 40.1 13.7

″818″ re-Designed Probe Set: SEQ ID NO: 5 CTAACACC G CCAATGTC818 SNP 5-A 17 30.2 42.4  7.8 SEQ ID NO: 6 CTAACACC A CCAATGTC818 SNP 5-D 17 46.7 36.5 10.2 SEQ ID NO: 7 AAAAA G GCGATACC G GG818 SNP 9-A 17 51.9 35.9 16.0 SEQ ID NO: 8 AAAAA T GCGATACC A GG818 SNP 9-D 17 46.8 24.6 22.2

The probe sets were then printed at 40 uM concentration in a MES/betainespotting buffer (300 mM MES at pH=4.5 and 1.5M betaine) on CorningGAPSII slides at two different times and immobilized by cross-linking byexposing the slides to 600mJ of UV. The first print run was forcomparison of the different syntheses of the “Tm Balanced” probe set(unmodified vs. 5′-ILinker modified) and spotted according to the probespot layout given in Table 1. The second print run was to evaluatehybridization of various 5′-ILinker modified probe designs (Tm Balancevs. 818) and spotted according to the probe spot layout given in Table2.

TABLE 1 Probe spot layout for different syntheses of the Tm Balancedprobe design. Tm SNP 5-A(unmod.) Tm SNP 5-D(unmod.) Tm SNP 9-A(unmod.)Tm SNP 9-D(unmod.) Tm SNP 5-A(unmod.) Tm SNP 5-D(unmod.) Tm SNP9-A(unmod.) Tm SNP 9-D(unmod.) Tm SNP 5-A(unmod.) Tm SNP 5-D(unmod.) TmSNP 9-A(unmod.) Tm SNP 9-D(unmod.) Tm SNP 5-A(unmod.) Tm SNP 5-D(unmod.)Tm SNP 9-A(unmod.) Tm SNP 9-D(unmod.) Tm SNP 5-A(unmod.) Tm SNP5-D(unmod.) Tm SNP 9-A(unmod.) Tm SNP 9-D(unmod.) Tm SNP 5-A(hydrazide)Tm SNP 5-D(hydrazide) Tm SNP 9-A(hydrazide) Tm SNP 9-D(hydrazide) Tm SNP5-A(hydrazide) Tm SNP 5-D(hydrazide) Tm SNP 9-A(hydrazide) Tm SNP9-D(hydrazide) Tm SNP 5-A(hydrazide) Tm SNP 5-D(hydrazide) Tm SNP9-A(hydrazide) Tm SNP 9-D(hydrazide) Tm SNP 5-A(hydrazide) Tm SNP5-D(hydrazide) Tm SNP 9-A(hydrazide) Tm SNP 9-D(hydrazide) Tm SNP5-A(hydrazide) Tm SNP 5-D(hydrazide) Tm SNP 9-A(hydrazide) Tm SNP9-D(hydrazide)

TABLE 2 Probe spot layout for different probe designs comparison. Tm SNP5-A(hydrazide) Tm SNP 5-D(hydrazide) Tm SNP 9-A(hydrazide) Tm SNP9-D(hydrazide) Tm SNP 5-A(hydrazide) Tm SNP 5-D(hydrazide) Tm SNP9-A(hydrazide) Tm SNP 9-D(hydrazide) Tm SNP 5-A(hydrazide) Tm SNP5-D(hydrazide) Tm SNP 9-A(hydrazide) Tm SNP 9-D(hydrazide) Tm SNP5-A(hydrazide) Tm SNP 5-D(hydrazide) Tm SNP 9-A(hydrazide) Tm SNP9-D(hydrazide) Tm SNP 5-A(hydrazide) Tm SNP 5-D(hydrazide) Tm SNP9-A(hydrazide) Tm SNP 9-D(hydrazide) 818 SNP 5-A(hydrazide) 818 SNP5-D(hydrazide) 818 SNP 9-A(hydrazide) 818 SNP 9-D(hydrazide) 818 SNP5-A(hydrazide) 818 SNP 5-D(hydrazide) 818 SNP 9-A(hydrazide) 818 SNP9-D(hydrazide) 818 SNP 5-A(hydrazide) 818 SNP 5-D(hydrazide) 818 SNP9-A(hydrazide) 818 SNP 9-D(hydrazide) 818 SNP 5-A(hydrazide) 818 SNP5-D(hydrazide) 818 SNP 9-A(hydrazide) 818 SNP 9-D(hydrazide) 818 SNP5-A(hydrazide) 818 SNP 5-D(hydrazide) 818 SNP 9-A(hydrazide) 818 SNP9-D(hydrazide)

Allele-specific synthetic hybridization targets were synthesized as5′-Cy3 oligonucleotides for A-allele detection and as 5′-Cy5oligonucleotides for D-allele detection. Sequences are shown below.

Allele-specific Hybridization Target Set: Name Len.SEQ ID NO: 9  Cy3-AGCTCTTGTGACATTGG C GGTGTTAGTGTAA Cy3 5-A 31SEQ ID NO: 10 Cy5-AGCTCTTGTGACATTGG T GGTGTTAGTGTAA Cy5 5-D 31SEQ ID NO: 11 Cy3-TTTTCCC C GGTATCGC C TTTTTAATTCTCAC Cy3 9-A 31SEQ ID NO: 12 Cy5-TTTTCCC T GGTATCGC A TTTTTAATTCTCAC Cy5 9-D 31

Allele-specific synthetic targets were hybridized at 50 nM concentrationin varying conditions with the cotton SNP array slides described above.After hybridization the slides were washed, dried, and scanned using theScanArray® 5000 (Perkin-Elmer) and laser (power/gain) settings of 69/69for the Tm Balance probe syntheses comparison (unmodified vs.5′-ILinker) and 68/68 for the different probe design comparison (TmBalanced vs. 818 sets).

Initial hybridizations using a traditional microarray hybridization/washbuffer composition and strategy (i.e.; overnight hybridization at 50° C.in 4.5×SSC and 0.25% Sarkosyl followed by three stringent washes at RT°C., consisting of 2×SSC/0.2% SDS for 5 mins for the 1^(st) wash, 1×SSCfor 2 mins for the 2^(nd) wash, and 0.2×SSC for 30 secs for the 3^(rd)wash) failed to discriminate between the cotton SNP alleles using the TmBalanced probe set. The Tm Balanced probes were also subjected to atemperature gradient hybridization strategy (FIG. 5A) to try to increaseallele discrimination using a TMAC-based hybridization buffer (i.e.;initial hybridization for 45 mins at 40° C. and subsequently increasedto 60° C. for an additional 1.5 hr in 37.5 mM Tris pH 8, 0.25% Sarkosyl,1 mM CTAB, and 1M TMAC followed by three stringent washes at RT° C.,consisting of 2×SSC/0.2% SDS for 5 mins for the 1^(st) wash, 2×SSC for 2mins for the 2^(nd) wash, and 0.2×SSC for 30 secs for the 3^(rd) wash)that also failed to adequately discriminate between the cotton SNPalleles. The 5′-ILinker modified Tm Balanced and 818 designed probeswere then hybridized with the new and improved hybridization buffer andwash strategy. The hybridization included a 2.25 hr hybridization at 50°C. in a 1× buffer comprising 37.5 mM Tris pH 8, 3 mM EDTA, 0.25%Sarkosyl, 0.4 mg/mL ovalbumin, 1 mM CTAB, 0.4 mg/mL Ficoll Type 400, 0.4mg/mL PVP-360, 2.5M TMAC, 10% Formamide, 10 ug/mL Cot-1 DNA followed bya first wash for 15 mins at 50° C. consisting of 2.5M TMAC and 0.2%Sarkosyl, a second wash for 1 min at RT° C. consisting of 2×SSC, and aquick dip (˜1-2 secs) in 0.2×SSC at RT° C. This hybridization and washstrategy showed improved SNP discrimination over that obtained using theprevious hybridization and wash conditions, particularly when combinedwith use of short 818 designed oligonucleotide probes (FIG. 5B).

The results, which are presented in FIG. 5, demonstrate the followingimportant points:

-   -   1) The Tm normalized probe set showed poor discrimination        between the tested cotton SNP alleles.    -   2) The new hybridization buffer and hybridization/wash protocols        developed improve ability of both the Tm normalized and short        818 probe sets to detect or discriminate between targets having        sequence differences that are not detectable using traditional        hybridization conditions.    -   3) Short 17mer “818” probes performed better than longer Tm        normalized probes. Using the new buffer/wash protocols        developed, discrimination was effectively 100%, giving        functional “On/Off” detection of alleles.    -   4) Use of the 5′ILinker modification was shown to significantly        improve signal intensity and therefore assay sensitivity without        affecting specificity.

Example 2 Preparation of Single-Stranded Labeled Targets Using EitherLambda or T7 Exonuclease Degradation of an Amplified Double-StrandedProduct.

The following example demonstrates a method to prepare labeledsingle-stranded targets from a double-stranded product of an exponentialPCR reaction. Lambda exonuclease and T7 exonuclease are both 5′ to 3′exonucleases that require and utilize double-stranded DNA (dsDNA) as asubstrate. Modifications to the 5′-end of one strand in the duplex thatconfer sensitivity to or protection against exonuclease activity resultin that strand being preferentially degraded by or protected fromexonuclease activity, while the opposite strand in the duplex ispreferentially protected or degraded. Thus, the exonuclease degradationreaction results in a single-stranded target suitable for hybridization.Nuclease sensitive or resistant modifications can be introduced by usingmodified primers in amplification reactions (e.g., PCR). Typically, oneprimer is modified and the second primer is unmodified.

The experiments described below determined that, contrary to previousreports, a 5′-phosphate group is not necessarily required for lambdaexonuclease activity. Further, certain fluorophores (e.g., Cy3) that areattached to the 5′-end of an oligo through standard phophoramiditechemistry confer resistance to lambda exonuclease digestion. Based onthe results reported herein, it was also discovered that T7 exonucleasedigestion can be blocked by a single phosphorothioate modifiedinternucleoside linkage, resulting in functionally complete protectionof a target DNA strand. The target DNA strand may contain another5′-modification, such as a Cy dye, linked to the target DNA through aphosphodiester or a phosphorothioate linkage. In addition, it wasdetermined that a phosphorothioate modified internucleoside linkage isnot required to block T7 exonuclease activity if the fluorophore (Cy3)is positioned between the second and third nucleotides from the 5′-endof the primer.

In this example, an oligonucleotide having an A/T 5′-endbase (5′-TCCT .. . -3′; sequence “a”) was employed and hybridized with a complementaryoligonucleotide sequence having a G/C 5′-endbase (5′-CCGA . . . -3′;sequence “b”) to form dsDNA. Different versions of each oligonucleotidesequence were synthesized to have various 5′-modifications, including5′-OH (unmodified), 5′-Phos, 5′-AmMC6, 5′-6FAM, or 5′-Cy3, with 0, 1, 2,or 3 phosphorothioate internucleoside bonds between the 5′-end bases. Inaddition, different versions of one of the oligonucleotide sequenceswere synthesized to include various internal Cy3 modifications with 0,1, or 2 phosphorothioate internucleoside bonds between the 5′-endnucleotides. Sequences are shown below, with “*” indicating a positionof phosphorothioate modified linkage.

SEQ ID NO: 13 T C C TCATTTCCAGAGAGAAGATCGG a- OH (0PS)SEQ ID NO: 14 T*C C TCATTTCCAGAGAGAAGATCGG a- OH (1PS+)SEQ ID NO: 15 T C*C TCATTTCCAGAGAGAAGATCGG a- OH (1PS+)SEQ ID NO: 16 T*C*C TCATTTCCAGAGAGAAGATCGG a- OH (2PS)SEQ ID NO: 17 T*C*C*TCATTTCCAGAGAGAAGATCGG a- OH (3PS)SEQ ID NO: 18 Phos-T C C TCATTTCCAGAGAGAAGATCGG a- Phos (0PS)SEQ ID NO: 19 AmMC6-TCCTCATTTCCAGAGAGAAGATCGG a-Am (0PS)SEQ ID NO: 20 6FAM-T C C TCATTTCCAGAGAGAAGATCGG a- FAM (0PS)SEQ ID NO: 21 Cy3-T C C TCATTTCCAGAGAGAAGATCGG a- Cy3 (0PS)SEQ ID NO: 22 TCATTTCCAGAGAGAAGATCGG a- Cy3 (1PS)SEQ ID NO: 23 Cy3-T C* C TCATTTCCAGAGAGAAGATCGG a- Cy3 (1PS+)SEQ ID NO: 24 Cy3-T*C8C TCATTTCCAGAGAGAAGATCGG a- Cy3 (2PS)SEQ ID NO: 25 Cy3-T*C*C* TCATTTCCAGAGAGAAGATCGG a- Cy3 (3PS)SEQ ID NO: 26 T-Cy3-C C TCATTTCCAGAGAGAAGATCGG a- Cy3+ (0PS)SEQ ID NO: 27 T*Cy3-C C TCATTTCCAGAGAGAAGATCGG a- Cy3+ (1PS)SEQ ID NO: 28 T-Cy3-C*C TCATTTCCAGAGAGAAGATCGG a- Cy3+ (1PS+)SEQ ID NO: 29 T*Cy3-C*C TCATTTCCAGAGAGAAGATCGG a- Cy3+ (2PS)SEQ ID NO: 30 T C-Cy3-C TCATTTCCAGAGAGAAGATCGG a- Cy3+2 (OPS)SEQ ID NO: 31 T*C-Cy3-C TCATTTCCAGAGAGAAGATCGG a- Cy3+2 (1PS)SEQ ID NO: 32 T C*Cy3-C TCATTTCCAGAGAGAAGATCGG a- Cy3+2 (1PS+)SEQ ID NO: 33 T*C*Cy3-C TCATTTCCAGAGAGAAGATCGG a- Cy3+2 (2PS)SEQ ID NO: 34 CCGATCTTCTCTCTGGAAATGAGGA b-OH (0PS)SEQ ID NO: 35 Phos-CCGATCTTCTCTCTGGAAATGAGGA b- Phos (0PS)SEQ ID NO: 36 AmMC6-CCGATCTTCTCTCTGGAAATGAGGA b- Am (0PS)SEQ ID NO: 37 6FAM-CCGATCTTCTCTCTGGAAATGAGGA b- FAM (0PS)

Each duplex reaction mixture includes an equal molar ratio of each oligorequired to form the duplex (oligo sequence “a” and oligo sequence “b”)in 1× NEBuffer 2 (50 mM NaCl from New England BioLabs). The duplexreaction mixture was heated for 5 minutes at 95° C. and allowed to coolto room temperature and sit overnight before the duplex wasover-digested relative to both time and enzyme units. Specifically, 40pmoles of each duplex was then digested overnight (15-18 hours) witheither 12.5 units of lambda exonuclease in the supplied buffer at 1×(New England BioLabs) or 25 units of T7 exonuclease in the suppliedbuffer at IX (New England BioLabs) at the appropriate temperature (37°C. or 25° C., respectively). All samples, along with a no enzyme controlfor each duplex reaction, were then separated on a 17% (19:1) nativeacrylamide gel in 1×TBE run for ˜1 hour at 25 Watts, stained withGelStar, and visualized on a UV transilluminator.

FIG. 6A shows that in this assay and under these gel conditions, theindividual single-stranded DNA oligos migrate differentially. Thevarious 5′-modifications (Cy3 and 6FAM) run as larger molecules relativeto the 5′-OH formulation of the same sequence (OH<Cy3<FAM). The “a”strand has a faster migration under these native acrylamide gelconditions and runs like a smaller molecule relative to the “b” strandmost likely due to the slight conformational change defined by thesequence composition differences between the two strands. Theconformational difference may, in fact, be caused by the stretch of A'sin the “b” strand that is not present in the “a” strand, which has beenpreviously demonstrated to cause bending in single-stranded DNA and slowthe migration in acrylamide gel electrophoresis. FIG. 6A also shows thatthe 5′-6FAM modification is sensitive to lambda exonuclease, but onlywhen it is attached to either an A or T at the 5′-end. In contrast, the5′-Cy3 modification confers lambda exonuclease resistance independent ofthe 5′-end sequence. This might be due to the fact that the cyanineclass of fluorescent dyes appears to increase the end-base stabilitythrough base-stacking interactions and thus interact with the nucleicacid in way that fluorescein class dyes do not.

FIG. 6B shows that the 5′-AmMC6 modification is also sensitive to lambdaexonuclease but only when it is attached to an A or T at the 5′-end. The5′-Phos modification is independent of the sequence and can initiate DNAdigestion regardless of the end-base composition. Surprisingly, thismodified composition is also partially sensitive to lambda exonucleaseeven when it is unmodified (i.e., 5′-OH). This sensitivity is affectedby digestion time and enzyme concentration, longer digestion time andhigher enzyme concentration in the digestion reaction yields morecomplete digestion of a DNA strand with an unmodified A/T 5′-end base.This observation is contrary to previous reports that a 5′-Phos isrequired for lambda exonuclease to initiate DNA digestion.

FIG. 6C shows that the 5′-Cy3 modification confers resistance to lambdaexonuclease that is independent of the 5′-end base composition. However,there does seem to be a slight sensitivity when the Cy3 modification isattached to a 5′-end with an A/T end-base pair. This seems to be mostheavily influenced by the stability of the opposite strand in the duplex(or dsDNA); that is, the 5′-Cy3 A/T end-base design is only slightlysensitive to lambda exonuclease when duplexed with a more stable strandsuch as one having a 5′-Cy3 G/C end-base. Therefore, generation of asingle-stranded target for microarray hybridization by lambdaexonuclease digestion of double stranded DNA could be enhanced by usinga primer having a 5′-cyanine dye attached to a G/C end-base to generatethe hybridization target strand and a primer having either a 5′-AmMC6 or5′-Phos attached to an A/T end-base to generate the non-target strand.

FIG. 6D shows that T7 exonuclease completely degrades DNA that isunmodified at its 5′-end (5′-OH). It also demonstrates that a singlephosphorothioate linkage can confer resistance to T7 exonuclease when itis placed in the penultimate internucleoside bond position (between thesecond and third nucleotides from the 5′-end) of an otherwise unmodifiedoligo (5′-OH). When the oligo also contains a 5′-Cy3 modification thesingle phosphorothioate linkage confers only partial T7 exonucleaseresistance when it is located in the ultimate internucleoside bondposition (between the first and second nucleotides from the 5′-end) andleaves the 5′-Cy3 modification intact. When the phosphorothioate linkageis located in the penultimate internucleoside bond position with theadditional 5′-Cy3 modification it appears that the resistant product ismissing the Cy3 dye. In addition, FIG. 6D shows that twophosphorothioate linkages placed between the first three 5′-endnucleotides is sufficient to confer functionally complete T7 exonucleaseprotection, whether the 5′-end of the PS modified strand is leftunmodified (5′-OH) or additionally modified with a fluorophore (5′-Cy3).The results using the 5′-Cy3 modified 2PS formulations demonstrate thatthe normal phosphodiester linkage between the 5′-Cy3 and the firstnucleotide is completely resistant to T7 exonuclease. There is also noevidence that the T7 exonuclease is sensitive to the differentphosphorothioate stereoisomers, as is the case with lambda exonuclease.

FIG. 6E demonstrates that T7 exonuclease resistance can be achievedutilizing a single phosphorothioate linkage in combination with a Cy3modification in this case, the Cy3 modification needs to be internal andplaced between the first and second nucleotides from the 5-end, whilethe phosphorothioate linkage is placed in the penultimateinternucleoside bond position. No additional resistance is achieved byadding a second PS linkage between the first nucleotide at the 5′-endand the internal Cy3, presumably because this is effectively theultimate internucleoside bond position that is already functionallymodified with the internal Cy3-modification (a detectable non-nucleosidemodified internucleoside linkage). In addition, FIG. 6E demonstratesthat a single stranded DNA having an internally positioned Cy3modification has T7 exonuclease resistance, even though it lacks a PSlinkage. This single-stranded DNA has a Cy3 modification between thesecond and third nucleotides from the 5′-end, i.e., the penultimateinternucleoside bond position. No additional resistance is achieved byadding one or more PS linkages. This is presumably because thepenultimate internucleoside bond is modified with the internal Cy3 inthe absence of an additional 5′-modification which is consistent withthe results in FIG. 6D using a single PS linkage in the penultimateinternucleoside bond position with an unmodified 5′-end (or 5′-OH).Therefore, a single-stranded target suitable for microarrayhybridization could be generated using T7 exonuclease to degrade thecomplementary strand of another strand made using a primer having aninternal dye (non-nucleoside based formulation such as Cy3) placed inthe penultimate internucleoside bond position or a primer with aninternal dye (non-nucleoside based formulation such as Cy3) placed inthe ultimate internucleoside bond position with a singlephosphorothioate linkage in the penultimate internucleoside bondposition.

Example 3

The following example illustrates the benefit of using single-strandednucleic acid targets over double-stranded nucleic acid targets formicroarray hybridizations, utilizing a synthetic SNP model system. Inaddition, this example demonstrates the greater sensitivity that can beachieved for sequence variation detection using various elements of themethod of the present invention.

Without consideration of the local sequence (or thermodynamic)environment of a SNP-site, the following short oligonucleotide arrayprobe set was synthesized as unmodified and as 5′-hydrazide modifiedoligonucleotides; both unpurified (desalted) and HPLC-purifiedpreparations were tested. The 17mer “818” design was employed.

p-101mer(C) SEQ ID NO: 38 TTCTGTGACTGGTGAGT p-101mer(G) SEQ ID NO: 39TTCTGTGAGTGGTGAGT p-101mer(A) SEQ ID NO: 40 TTCTGTGAATGGTGAGTp-101mer(T) SEQ ID NO: 41 TTCTGTGATTGGTGAGTUnderlined bases indicate the synthetic SNP site.

For this example, oligonucleotides were printed at 40 uM in 1× OligoSpotting Buffer, OSB, (Integrated DNA Technologies, Inc.) on CorningGAPSII slides and immobilized by UV cross-linking with 600mJ using theStrataLinker 2400 (Stratagene). The 5′-hydrazide modified array probeswere spotted in FIG. 7 according to the probe spot layout given in Table3, while the unmodified desalted and 5′-hydrazide modified desaltedarray probes were spotted in FIG. 8 according to the probe spot layoutgiven in Table 4.

TABLE 3 Probe spot layout of the different purity 5′-hydrazide synthesesin FIG. 7. p-101mer(T) - HPLC p-101mer(T) - HPLC p-101mer(T) - HPLCp-101mer(T) - HPLC p-101mer(A) - HPLC p-101mer(A) - HPLC p-101mer(A) -HPLC p-101mer(A) - HPLC p-101mer(C) - HPLC p-101mer(C) - HPLCp-101mer(C) - HPLC p-101mer(C) - HPLC p-101mer(G) - HPLC p-101mer(G) -HPLC p-101mer(G) - HPLC p-101mer(G) - HPLC p-101mer(T) - desaltp-101mer(T) - desalt p-101mer(T) - desalt p-101mer(T) - desaltp-101mer(A) - desalt p-101mer(A) - desalt p-101mer(A) - desaltp-101mer(A) - desalt p-101mer(C) - desalt p-101mer(C) - desaltp-101mer(C) - desalt p-101mer(C) - desalt p-101mer(G) - desaltp-101mer(G) - desalt p-101mer(G) - desalt p-101mer(G) - desalt

TABLE 4 Probe spot layout of the desalted array probes in FIG. 8.p-101mer(T) p-101mer(T) p-101mer(T) p-101mer(T) (unmodified)(unmodified) (unmodified) (unmodified) p-101mer(A) p-101mer(A)p-101mer(A) p-101mer(A) (unmodified) (unmodified) (unmodified)(unmodified) OSB OSB OSB OSB p-101mer(C) p-101mer(C) p-101mer(C)p-101mer(C) (unmodified) (unmodified) (unmodified) (unmodified)p-101mer(G) p-101mer(G) p-101mer(G) p-101mer(G) (unmodified)(unmodified) (unmodified) (unmodified) p-101mer(T) p-101mer(T)p-101mer(T) p-101mer(T) (5′-hydrazide) (5′-hydrazide) (5′-hydrazide)(5′-hydrazide) p-101mer(A) p-101mer(A) p-101mer(A) p-101mer(A)(5′-hydrazide) (5′-hydrazide) (5′-hydrazide) (5′-hydrazide) OSB OSB OSBOSB p-101mer(C) p-101mer(C) p-101mer(C) p-101mer(C) (5′-hydrazide)(5′-hydrazide) (5′-hydrazide) (5′-hydrazide) p-101mer(G) p-101mer(G)p-101mer(G) p-101mer(G) (5′-hydrazide) (5′-hydrazide) (5′-hydrazide)(5′-hydrazide) Guide Guide Guide Guide Light Light Light Light Note:Guide light represents a control oligonucleotide that is Cy3 dye labeledfor the purpose of image orientation and does not participate inhybridization reactions.

The following 101mer unmodified oligonucleotides were synthesized foruse as synthetic templates to generate single-stranded vs.double-stranded labeled targets by PCR amplification. These synthetictemplates represent the target strand that is complementary to the arrayprobe sequences above:

SEQ ID GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAG t-101mer(G) NO:42TCACAGAACAGATGGTGCAGAGGGCCATGAAGGACCTGACCTAT GCCTCCCTGTGC SEQ IDGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAC t-101mer(C) NO: 43TCACAGAACAGATGGTGCAGAGGGCCATGAAGGACCTGACCTAT GCCTCCCTGTGC SEQ IDGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAT t-101mer(T) NO: 44TCACAGAACAGATGGTGCAGAGGGCCATGAAGGACCTGACCTAT GCCTCCCTGTGC SEQ IDGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAA t-101mer(A) NO: 45TCACAGAACAGATGGTGCAGAGGGCCATGAAGGACCTGACCTAT GCCTCCCTGTGC Underlinedbases indicate the synthetic SNP site.

Double-stranded target material was generated via exponential PCRmethods from ˜2.0E+05 copies of synthetic 101mer template (total) per 50μL reaction volume containing 200 nM each of a forward primer(Cy3-G*C*CGCATACACTATTCTCAG (SEQ ID NO:46); * indicates position of aphosphorothioate linkage) and a reverse primer (GCACAGGGAGGCATAGGT) (SEQID NO:47); utilizing the following cycling conditions: initial melt @95°C. for 9.5 mins followed by 35 cycles of 95° C. for 30 secs, 59° C. for20 secs, 72° C. for 30 secs.

Single-stranded hybridization target material was generated by digesting20 μL of the PCR reaction above in a 60 μL digestion volume using 10units of T7 exonuclease and the supplied exonuclease digestion buffer atIX (New England BioLabs) for 2 hours at room temperature, using themethods described in example 2 above. After digestion, the targetmaterial was column-purified using the Promega Chip Shot membrane andprotocol, followed by lyophilization of the eluted target material. Thedouble-stranded hybridization target material (used in FIG. 7) wasgenerated by setting up a “mock” digestion using 20 μL of the same PCRreaction used for the single-stranded target generation (i.e.; withoutthe T7 exonuclease) immediately followed by the ChipShot membranepurification and lyophilization.

The dried target was resuspended in 1×SNP Hybridization Buffer andhybridized to the Microarray slide for 2.25 hours at 50° C., followed bya 15 min wash at 50° C. in 1×SNP Wash Buffer 1, a 1 min wash at RT° C.in 2×SSC, and a quick dip (1-2 secs.) in 0.2×SSC. For I×SNP Hyb Buffer,the composition is: 37.5 mM Tris pH 8, 3 mM EDTA, 0.25% Sarkosyl, 0.4mg/mL Ovalbumin, 1 mM CTAB, O.4 mg/mL Ficoll Type 400, 0.4 mg/mLPVP-360, 2.5M TMAC (tetramethyl ammonium chloride), 10% Formamide, 10ug/mL Cot-1 DNA. The composition of the 1×SNP Wash Buffer 1 is: 2.5MTMAC, 0.2% Sarkosyl.

After washing the slides were dried and scanned using a ScanArray® 5000(Perkin-Elmer) with laser (power/gain) settings of 89/89 for FIG. 7 and85/85 for FIG. 8. After the specific-target hybridization for the arraysin FIG. 7, the slides were re-hybridized with SpotQC (Integrated DNATechnologies, Inc.) and re-visualized using the ScanArray® 5000(Perkin-Elmer) to help orient the array images from the single-strandedand double-stranded target hybridizations relative to the print layout.

FIG. 7 demonstrates that improved sensitivity is achieved using asingle-stranded hybridization target compared to the same hybridizationtarget when it is double-stranded. In addition, the results in FIG. 7show that the hydrazide modification can also attach to theamine-surface of the Coming GAPSII slides. The efficiency of thisattachment is sufficient that the typical post-synthesis oligopurification (i.e.; HPLC purification) is not necessary as it is withmany other covalent attachment strategies known in the art. The resultsshown in FIG. 8 further support this conclusion since the hybridizationsignals from the 5′-hydrazide modified unpurified desalted synthesisoligo probes are significantly stronger than the hybridization signalsfrom their unmodified probe versions when hybridized to the same targetunder the same conditions. In addition, these results demonstratecomplete specificity resulting in perfect discrimination of just asingle nucleotide difference even in this example that uses a G/Tmismatch, which is the most unfavorable of all possible mismatchbase-pairings for discrimination. Thus, the combination of probe design,probe modification, hybridization buffer, and wash conditions andprotocols are sufficient to confer both increased sensitivity andselectivity.

Example 4 Improved Buffer for Spotting Nucleic Acid Probes to an EpoxideSurface

This example describes a spotting buffer for attaching nucleic acidprobes on epoxide surfaces. The spotting buffer is compatible with useof unmodified, amino-modified, and hydrazide modified nucleic acids. Acombination of monobasic sodium phosphate (low pH), Nonidet P-40(NP-40), and ethylene glycol were used for spotting oligo probes onto anepoxide-surface microarray slide. For a 1× formulation, the compositionof the Epoxide Spotting Buffer is: 300 mM sodium phosphate (monobasic);0.01% NP-40; 45% ethylene glycol. The ranges can be from 1 mM to IMsodium phosphate (monobasic), 0.001% to 1% NP-40, and from 10% to 90%ethylene glycol. The pH range can be from 4 to 8.

Conventional spotting solutions that were considered compatible with orspecifically made for use on epoxide slides produce probe spots that areeither too small for analysis or too large to allow high-densitymicroarray printing. In both instances, the hybridization signal isnon-uniform. In addition, the “large” spot phenotype often results inspot merger, which is dependent on the oligonucleotide composition,making the merged probe areas unpredictable and useless for microarrayexperiments. This problem is illustrated in FIG. 9, which comparesdifferent epoxide spotting solutions. Oligonucleotide probes (standarddesalted 41mers) were spotted at 40 μM concentration on an epoxide slide(Corning) using the above epoxide spotting buffer formulation (ESB),3×SSC, and three different commercially available spotting solutionsmarketed as either “specifically formulated for use on”, or “compatiblewith” the epoxide slide surface. The sub-arrays were then hybridizedwith complementary Cy3™-labeled oligo targets and scanned, using theScanArray® 5000 (Perkin-Elmer) at 62/62 laser (power/gain) settings. Theneed for additives to increase the spot size to something larger than a“pin prick” is also illustrated in FIG. 9 by the sub-panel where theoligo probes are spotted in a solution containing just sodium chlorideand sodium citrate (3×SSC). These spotting problems, or spot morphologyissues, are primarily due to the incompatibility of the additives thatare typically used with the epoxide surface, which is typicallymanufactured by the silanization of glass slides with3-glycidoxypropyltrimethoxysilane. Since the epoxide surface is notcharged (i.e.; non-ionic), the use of ionic, or polar, detergents(especially anionic such as SDS and Sarkosyl) result in non-uniformhybridization spot signals characterized by either “rings” of signalintensity or random “crystal-like” patterns. In addition, the spot sizeis difficult to control with these types of detergents since smallamounts, or small changes in concentration, dramatically affect theprinted probe spot size. Consequently, very little variation from the“best” amount typically results in altered spot size/morphology.Evaporation of the water that typically comprises the remaining volumeof the spotting solution causes increased detergent concentrationswithin the spotted material resulting in oversized, merged probe spots.

The combination of these ethylene oxide based reagents for spottingoligo probes onto an ethylene oxide based surface allows for greatercontrol of printed spot size and the ability to fine tune the spot sizeby the addition of easily measured amounts, as illustrated in FIG. 10.The oligonucleotide probes (desalted 70mers) were spotted at 40 μMconcentration on an epoxide slide (Corning) using the above epoxidespotting buffer formulation supplemented with varying concentrations ofNP-40. The sub-arrays were then hybridized with IDT's Cy3™-SpotQC andscanned, using the ScanArray® 5000 (Perkin-Elmer) at 69/69 (optimizedfor 50% GC probe) & 78/78 (optimized for 38.6% GC probe) laser(power/gain) settings.

Increasing the pH of the spotting solution by either titrating in sodiumhydroxide (NaOH) or by changing the source of sodium (3×SSC) results indecreased hybridization signal, suggesting decreased oligo probeattachment density within the probe spot, and increased probe spot size.In FIG. 10, oligonucleotide probes (unpurified 41 mers) were spotted at40 uM concentration on an epoxide slide (Coming). using differentspotting solution formulations that varied by source of sodium (300 mMsodium phosphate, monobasic vs. 3×SSC) and pH. The sub-arrays were thenhybridized with complementary Cy3™-labeled oligo targets and scanned,using the ScanArray® 5000 (Perkin-Elmer) at 60/60 laser (power/gain)settings. The lower % GC results in smaller diameter printed probespots.

Example 5 Functional Example of a Genetic Variation Discrimination Assay

The following example illustrates the design of a generic variationdiscrimination assay utilizing both the “traditional” microarray formatand a “reverse” microarray format. The Let7 miRNA family was used as amodel system since it provides different degrees of sequence variationwithin a single family of highly similar and biologically relevantsequences. In addition, each element of this model can be mimicked usingsynthetic oligonucleotides and these mimics can be used to simulatebiological environments with known expression levels and patterns tomore effectively and efficiently characterize the specificity, as wellas the sensitivity, of this improved microarray system.

In an assay with an actual sample obtained from a biological source, thesmall RNA fragments would first be isolated using a product, or kit,such as the Ambion® mirVana column. A 3′ linker would then be attachedto the small RNA, such as is used in the miRCat cloning kit (IntegratedDNA Technologies, Inc.), to provide a reverse transcription (RT) primingsite. After post-linkering purification, the target strands are modifiedwith a 5′-fluorophore using a fluorophore-labeled RT primer. Thus asingle-stranded hybridization target is obtained which can be hybridizedagainst short (16-17mer) 5′-hydrazided modified oligonucleotide probesimmobilized on an epoxide slide surface in a “traditional” microarrayformat. The target strands can also be modified with a 5′-hydrazidegroup using a hydrazide-modified RT primer for immobilization on anepoxide slide surface and subsequently hybridized with afluorophore-labeled short (16-17mer) synthetic oligo probe set. In thisexample, results of Let7 model hybridizations using a “traditional” DNAmicroarray format are illustrated in FIG. 11, and those using a“reverse” DNA microarray format are illustrated in FIG. 12.

There are twelve Let7 microRNA loci in the human genome that resolveinto nine discrete mature Let7 miRNA sequences. There are three lociwhere hsa-let-7a occurs (hsa-let-7a-1, -2, and -3) and two loci forhsa-let-7f (hsa-let-7f-1 and -2).

The following 16-mer (in bold) and 17-mer Human Let7 oligo probe setswere selected using miRBase (Release 8.0) and synthesized as either5′-hydrazide modified oligo probes for use in the “traditional” arrayformat or 5′-Cy3 modifiedoligo probes for use in the “reverse” arrayformat.

SEQ ID NO: 48 GTAGTAGGTTGTATAGT Let 7a (17mer) SEQ ID NO: 49 TAGTAGGTTGTATAGT Let 7a (16mer) SEQ ID NO: 50 GTAGTAGGTTGTGTGGTLet 7b (17mer) SEQ ID NO: 51  TAGTAGGTTGTGTGGT Let 7b (16mer)SEQ ID NO: 52 GTAGTAGGTTGTATGGT Let 7c (17mer) SEQ ID NO: 53 TAGTAGGTTGTATGGT Let 7c (17mer) SEQ ID NO: 54 GTAGTAGGTTGCATAGTLet 7d (17mer) SEQ ID NO: 55  TAGTAGGTTGCATAGT Let 7d (16mer)SEQ ID NO: 56 GTAGGAGGTTGTATAGT Let 7e (17mer) SEQ ID NO: 57 TAGGAGGTTGTATAGT Let 7e (16mer) SEQ ID NO: 58 GTAGTAGATTGTATAGTLet 7f (17mer) SEQ ID NO: 59  TAGTAGATTGTATAGT Let 7f (16mer)SEQ ID NO: 60 GTAGTAGTTTGTACAGT Let 7g (17mer) SEQ ID NO: 61 TAGTAGTTTGTACAGT Let 7g (16mer) SEQ ID NO: 62 GTAGTAGTTTGTGCTGTLet 7i (17mer) SE0 ID NO: 63  TAGTAGTTTGTGCTGT Let 7i (17mer)SEQ ID NO: 64 GTAGTAAGTTGTATTGT miR-78 (17mer) SEQ ID NO: 65 TAGTAAGTTGTATTGT miR-98 (17mer) Underlined bases indicate the variationrelative to 7a.

The Let-7 target “mimics” and a partially “randomized” miRCat-pooltarget mimic were synthesized as 5′-Cy3 oligos for hybridization withthe “traditional” array format or as 5′-hydrazide modified oligos forimmobilization on an epoxide slide surface with the “reverse” arrayformat. The partially “randomized” miRCat-pool target mimic was used tosimulate the post-reverse transcription “background complexity” thatmight be expected if using real RNA isolated from a sample (or tissue)of interest.

SEQ ID NO: 66 GTCCTTGGTGCCCGAGTGTAACTATACAACCTACTACCTCA Let-7aSEQ ID NO: 67 GTCCTTGGTGCCCGAGTGTAACCACACAACCTACTACCTCA Let-7bSEQ ID NO: 68 GTCCTTGGTGCCCGAGTGTAACCATACAACCTACCTCA Let-7c SEQ ID NO: 69 GTCCTTGGTGCCCGAGTGTACTATGCAACCTACTACCTC Let-7dSEQ ID NO: 70 GTCCTTGGTGCCCGAGTGTACTATCAACCTCCTACCTCA Let-7eSEQ ID NO: 71 GTCCTTGGTGCCCGAGTGTAACTATACAATCTACTACCTA Let-7fSEQ ID NO: 72 GTCCTTGGTGCCCGAGTGTACTGTAAACTACCTA Let-7g SEQ ID NO: 73GTCCTTGGTGCCCGAGTGTACAGCACAAACTACTACCTCA Let-7i SEQ ID NO: 74GTCCTTGGTGCCCAGTGTAACAATACAACTTACTCCTCA miR9B SEQ ID NO: 75GTCCTTGGTGCCCGAGTGTNNNNNNNNNNNNNNNNNNNNNNN miRCat- pool

For the “traditional” array format example, the 16mer and 17mer5′-hydrazide modified oligo probe sets were each printed at a 40 μMconcentration in 1× epoxide spotting buffer (see Example 4 for buffercomposition) and immobilized on epoxide slides (Corning), according tothe probe spot layout given in Table 5.

TABLE 5 “Traditional” Array Format Probe Spot Layout Guide Guide LightLight Let-7a Let-7a Let-7b Let-7b Let-7c Let-7c (17mer) (17mer) (17mer)(17mer) (17mer) (17mer) Let-7d Let-7d Let-7e Let-7e Let-7f Let-7f(17mer) (17mer) (17mer) (17mer) (17mer) (17mer) Let-7g Let-7g Let-7iLet-7i miR98 miR98 (17mer) (17mer) (17mer) (17mer) (17mer) (17mer)Let-7a Let-7a Let-7b Let-7b Let-7c Let-7c (16mer) (16mer) (16mer)(16mer) (16mer) (16mer) Let-7d Let-7d Let-7e Let-7e Let-7f Let-7f(16mer) (16mer) (16mer) (16mer) (16mer) (16mer) Let-7g Let-7g Let-7iLet-7i miR98 miR98 (16mer) (16mer) (16mer) (16mer) (16mer) (16mer) GuideGuide Light Light

The Cy3-labeled target mimics were then hybridized to the “traditional”array format slide at 50° C. for 2.25 hours. One slide was hybridizedwith a pool of all nine Cy3-labeled Let7 target mimics (the positivecontrol hyb) at a total concentration of 10 nM; each individual Let7target mimic was represented at 1.11 nM in this hybridization mix. Asecond slide was hybridized with just the Cy3-labeled miRCat-pool targetmimic (the negative control hyb) at a concentration of 1O0 nM. Otherslides were then hybridized with a different specific Cy3-Let7 targetmix with a total target concentration of 10 nM; each specificLet7-target mix contained just a single Cy3-Let7 target at 1.11 nMconcentration within the general Cy3-miRCat-pool target background.After hybridization the slides were washed, dried, and scanned using thesame laser power/gain settings. FIG. 11 demonstrates that all probespots are easily detected when the appropriate hybridization target ispresent at 1.11 nM and that the 17mer probe set has greaterhybridization signal strength compared to the 16mer probe set. It alsodemonstrates the specificity of this microarray system since none of theprobe spots are detected when hybridized with an unrelated targetsequence (the negative control hybridized with the Cy3-miRCat-pooltarget), and only the specific probe spots are easily visualized whenhybridized with the appropriate specific Let7-target mix (example withCy3-Let-7a). Only minimal cross-hybridization is detected with thespecific Let7-target hybridization mixes which is limited to the mostclosely related sequences within the Let7 family and is at the detectionthreshold, or lowest sensitivity limit of the assay, of detection whenthe specific probe spot hybridization signal is well above the signalsaturation limit comparison and alignment of the Let-7a target with thecross-hybridizing probe spots Let-7c and Let-7e, which are barelydetectable in this example figure is below. A summary of thehybridization results from all nine independent specific Let7-targetmixes is given in Table 6.

Let-7a Target ACTCCATCATCCAACATATCAATGTGAGCCCGTGGTTCCTG/5Cy3/SEQ ID NO: 66 in reverse orientation, 3′ to 5′) Let-7c Probe/5ILink12/GTAGTAGGTTGTAT G GT (SEQ ID NO: 52)(T/G mismatch with 14 contiguous nt) Let-7e Probe /5ILink12/GTAGTAG GTTGTATGGT (SEQ ID NO: 56) (A/G mismatch with 12 contiguous nt)

TABLE 6 Specific Let7-target Hybridization Results Probe Target: 7a 7b7c 7d 7e 7f 7g 7i miR98 7a +++++ − − + − − − − − 7b − +++++ − − − − − −− 7c + − +++++ − − − − − − 7d − − − +++++ − − − − − 7e + − − + +++++ − −− − 7f − − − − − ++++ − − − 7g − − − − − − +++++ − − 7i − − − − − − −+++++ − miR98 − − − − − − − − +++++

For the “reverse” array format example, the individual 5′-hydrazidemodified Let7 target mimic sequences were sorted into various mixes(detailed below) and subsequently mixed with the 5′-hydrazide modifiedmiRCat-pool target mimic to simulate the genetic complexity that onemight expect from using a sample of interest with these defined Let7compositions. Each mix was printed at a 40 μM total oligo concentration(the individual Let7 sequences represent molecular ratios from ˜1:10 to˜1:10,000, relative to the total molecule composition) in 17× epoxidespotting buffer (see Example 4 for buffer composition) and immobilizedon epoxide slides (Corning), according to the probe spot layout given inTable 7.

TABLE 7 “Reverse” Array Format Probe Spot Layout Mix 11 Mix 11 Mix 12Mix 12 Buffer Buffer Guide Guide (1) (1) (1) (1) (N) (N) Light Light Mix7 Mix 7 Mix 8 Mix 8 Mix 9 Mix 9 Mix 10 Mix 10 (N) (N) (N) (N) (C) (C)(1) (1) Mix 3 Mix 3 Mix 4 Mix 4 Mix 5 Mix 5 Mix 6 Mix 6 (N) (N) (N) (N)(2) (2) (C) (C) Guide Guide Buffer Buffer Mix 1 Mix 1 Mix 2 Mix 2 LightLight (N) (N) (1) (1) (C) (C) Buffer = spotting buffer only Mix 1 =minus 7c, 7d Mix 2 = minus 7a, 7d Mix 3 = minus 7a, 7c Mix 4 = minus 7a,7c, 7d & 2X 7e Mix 5 = minus 7b, 7c, 7d & 2X 7a Mix 6 = minus 7a, 7d, 7e& 2X 7b Mix 7 = minus 7a, 7c, 7f & 2X 7g Mix 8 = minus 7a, 7c, 7d, 7g &2X 7f, 7i Mix 9 = minus 7a, 7b, 7f, 7i & 2X 7d, 98 Mix 10 = minus 7b,7e, 7g, 98 & 2X 7c, 7f Mix 11 = minus 7b, 7c, 98 & 2X 7d Mix 12 = minus7d, 7i (N) = expected negative spot since it lacks 7a target and 7c (1)= expected positive signal with 7a target (2) = expected 2X signal sinceit contains twice the amount of 7a target (C) = possiblecross-hybridization since 7c is present

The Cy3-labeled Let-7a 16mer oligo probe was hybridized to the “reverse”array format slide at 10 nM concentration at 50° C. for 2.25 hours. Fivearray sets representing five print, or spotting, dilutions were tested(see FIG. 12).

FIG. 12 shows that the expected signal pattern is clearly visible at the1/1,000 ratio and right at the lower limits of detection at the 1/10,000ratio, demonstrating a high degree of sensitivity of this array systemeven in the “reverse” array format. This level of sensitivity is similarto that achieved with the “traditional” array format. The specificity isalso maintained with no evidence of Let-7a cross-reactive hybridizationsignal with the probe spots containing Let-7c.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. An array comprising a surface comprising epoxidemoieties and a plurality of oligonucleotides each comprising a 5′hydrazide linker attached to the surface of the array through a bondformed between the linker and an epoxide moiety.
 2. A method of forminga microarray on a surface comprising epoxide moieties, comprising:depositing a plurality of samples onto the surface in discrete domains,each sample comprising a spotting buffer and an oligonucleotidecomprising a 5′ hydrazide linker, under conditions that allow formationof a bond between the hydrazide linker and the epoxide moiety.
 3. Themethod of claim 2, wherein the spotting buffer has a pH of less thanabout 8.5 and comprises monobasic sodium phosphate, an ethylene oxidebased nonionic detergent, and ethylene glycol.
 4. The method of claim 3,wherein the nonionic detergent is Nonidet P-40.
 5. The method of claim3, wherein the pH is between about 4.0 and about 8.0.
 6. The method ofclaim 3, wherein the pH is between about 4.5 and about 5.5.